In many applications it is desirable to enrich, or alternatively deplete, certain cell populations in a biological sample. For example, the separation of specific cell types from peripheral blood, bone marrow, spleen, thymus and fetal liver is key to research in the fields of hematology, immunology and oncology, as well as diagnostics and therapy for certain malignancies and immune disorders.
Investigation of cellular, molecular and biochemical processes requires analysis of certain cell types in isolation. For example purified populations of immune cells such as T cells and antigen presenting cells are necessary for the study of immune function and are used in immunotherapy. Most cell separation techniques require that the input sample be a single cell suspension. For this reason, blood has historically been the most common tissue used for cell separations. Numerous techniques have been used to isolate T cell subsets, B cells, basophils, NK cells and dendritic cells from blood for these investigations.
More recently, enzymatic digestion methods have been developed to dissociate tissues from solid organs into single cell suspensions, permitting distinct cell types to be isolated. This is of particular benefit to the study of pluripotent stem cells and tissue-specific stem cells from adults. The rapidly growing field of stem cell research is spurred by the potential of these cells to repair diseased or damaged tissues. Bone marrow (hematopoietic) stem cells were the first adult stem cells to be purified and used clinically and the therapeutic potential of hematopoietic stem cells is now well documented. Transplantation of hematopoietic cells from peripheral blood and/or bone marrow is increasingly used in combination with high-dose chemo- and/or radiotherapy for the treatment of a variety of disorders including malignant, non-malignant and genetic disorders. Very few cells in such transplants are capable of long-term hematopoietic reconstitution, and thus there is a strong stimulus to develop techniques for purification of hematopoietic stem cells. Furthermore, serious complications and indeed the success of a transplant procedure is to a large degree dependent on the effectiveness of the procedures that are used for the removal of cells in the transplant that pose a risk to the transplant recipient. Such cells include T lymphocytes that are responsible for graft versus host disease (GVHD) in allogeneic grafts, and tumor cells in autologous transplants that may cause recurrence of the malignant growth. It is also important to debulk the graft by removing unnecessary cells and thus reducing the volume of cyropreservant to be infused.
In certain instances it is desirable to remove or deplete tumor cells from a biological sample, for example in bone marrow transplants. Epithelial cancers of the bronchi, mammary ducts and the gastrointestinal and urogenital tracts represent a major group of solid tumors seen today. Micrometastatic tumor cell migration is thought to be an important prognostic factor for patients with epithelial cancer (Braun et al., 2000; Vaughan et al., 1990). The ability to detect such metastatic cells is limited by the effectiveness of tissue or fluid sampling and the sensitivity of tumor detection methods. A technique to enrich circulating epithelial tumor cells in blood samples would increase the ability to detect metastatic disease and facilitate the study of such rare cells to determine the biological changes which enable spread of the disease.
Hematopoietic cells and immune cells have been separated on the basis of physical characteristics such as density and on the basis of susceptibility to certain pharmacological agents which kill cycling cells. The advent of monoclonal antibodies against cell surface antigens has greatly expanded the potential to distinguish and separate distinct cell types. There are two basic conceptual approaches to separating cell populations from blood and related cell suspensions using monoclonal antibodies. They differ in whether it is the desired or undesired cells which are distinguished/labeled with the antibody(s).
In positive selection techniques the desired cells are labeled with antibodies and removed from the remaining unlabeled/unwanted cells. In negative selection, the unwanted cells are labeled and removed. Antibody/complement treatment and the use of immunotoxins are negative selection techniques, but Fluorescence Activated Cell Sorting (FACS) and most batch-wise immunoadsorption techniques can be adapted to both positive and negative selection. In immunoadsorption techniques, cells are selected with monoclonal antibodies and preferentially bound to a surface which can be removed from the remainder of the cells e.g. column of beads, flask, magnetic particles. Immunoadsorption techniques have won favor clinically and in research because they maintain the high specificity of cell targeting with monoclonal antibodies, but unlike FACS, they can be scaled up to directly process the large numbers of cells in a clinical harvest and they avoid the dangers of using cytotoxic reagents such as immunotoxins and complement.
Magnetic separation is a process used to selectively retain magnetic materials within a vessel, such as a centrifuge tube or column, in a magnetic field gradient. Targets of interest, such as specific biological cells, can be magnetically labelled by binding of magnetic particles to the surface of the targets through specific interactions including immuno-affinity interactions. Other useful interactions include drug-drug receptor, antibody-antigen, hormone-hormone receptor, growth factor-growth factor receptor, carbohydrate-lectin, nucleic acid sequence-complementary nucleic acid sequence, enzyme-cofactor or enzyme-inhibitor binding. The suspension, containing the targets of interest within a suitable vessel, is then exposed to magnetic field gradients of sufficient strength to separate the targets from other entities in the suspension. The vessel can then be washed with a suitable fluid to remove the unlabeled entities, resulting in a purified suspension of the targets of interest.
The majority of magnetic labeling systems use superparamagnetic particles with antibodies or streptavidin covalently bound to their surface. In cell separation applications these particles can be used for either positive selection, where the cells of interest are magnetically labelled, or negative selection where the majority of undesired cells are magnetically labelled. Magnetic separation applications where the targets of interest are proteins or nucleic acids would be considered positive selection approaches since the entity of interest is typically captured on the magnetic particle. The diameter of the particle used varies widely from about 50-100 nm for MACS particles (Miltenyi Biotec) and StemSep™ colloid (StemCell Technologies), through 150 nm-1.5 μm for EasySep™ (StemCell Technologies) and Imag (BD Biosciences) particles and 1 to 4.2 μm for Dynabeads (Invitrogen). The type of particle used is influenced by the magnet technology employed to separate the labelled entities.
There are two important classes of magnetic separation technologies, both of which, for convenience and practical reasons use permanent magnets as opposed to electromagnets. The first class is column-based high-gradient-magnetic-field separation technology that uses small, weakly magnetic particles to label the targets of interest, and separates these targets in a column filled with a magnetizable matrix. Very high gradients are generated close to the surface of the matrix elements when a magnetic field is applied to the column. The high gradients are necessary to separate targets labelled with these relatively weakly magnetic particles. The second class is tube-based technology that uses more strongly magnetic particles to label the targets of interest. These targets of interest are then separated within a centrifuge-type tube by magnetic field gradients generated by a magnet outside the tube. This method has the advantage that it does not rely on a magnetizable matrix to generate the gradients and therefore does not required an expensive disposable column or a reusable column with an inconvenient cleaning and decontamination procedure.
Once placed within the magnet, targeted cells migrate toward the region or regions of highest magnetic field strength and are retained within the magnetic field while the unlabeled cells are drawn off. The targeted cells can then be collected and used in the desired application after removal from the magnetic field. In the event that negative selection is required, the unlabeled cells are drawn off and can be utilized for a variety of applications such as cell sorting.
The EasySep labeling method uses a tetrameric antibody complex (TAC) (U.S. Pat. No. 4,868,109). TACs are comprised of two mouse IgG1 monoclonal antibodies held in tetrameric array by two rat anti-mouse IgG1 antibody molecules. EasySep™ reagents cross-link magnetizable particles to cells of interest using TAC where one mouse antibody targets the particles and the other targets surface markers on the cells of interest. The EasySep™ magnet then separates the magnetically labeled cells from non-labeled cells within a standard centrifuge tube.
After magnetic labeling and initial magnetic separation, it is necessary to wash away non-specifically separated cells to attain the desirable level of fractionation of the sample into labeled and unlabeled cells. In tube-based systems such as Dynal® and EasySep®, the separation vessel is typically a standard centrifuge tube held in a magnetic field. To obtain high purity of the target cells in positive selection applications, a sequence of batch wash steps is used to remove unlabelled cells from the separation tube, where the supernatant containing the majority of unlabeled cells is removed from the separation vessel and then the retained cells are resuspended and re-separated a number of times as required.
Larger magnetic particles (>0.5 μm) have the advantage that separation times can be lower, and weaker magnetic fields can be used. For example, Dynal particles of 1.0, 2.5 and 4.2 μm diameter are separated by a simple side-pull magnet with a peak magnetic field strength of about 0.3 T. Polysciences BioMag Plus particles of 1.0 μm diameter may be separated in 96-well plates using a pull-down plate magnet such as Dexter Magnetics LifeSep 96F. However, it is well known that larger magnetic particles also strongly affect the light scatter signature of the cells to which they are bound when analyzed by FACS. Larger particles bound to cells affect both the forward scatter and side scatter signal, with the side scatter signal being more severely affected. The forward scatter signal correlates to cell size, while the side scatter signal correlates to granularity. Forward and side scatter signals are important component of FACS analysis and distortion of these signals can significantly impair the interpretation of the results. A large magnetic particle that, when bound to cells, does not exhibit this effect on the light scattering property of the cells would be of high utility for magnetic cell separation protocols. Larger particles also interfere with visible light microscopy of the cells by changing the apparent cell morphology. A large particle that, when bound to cells, does not appreciably change the apparent morphology of the cells when viewed by visible light microscopy would also be of high utility for magnetic cell separation protocols.